Gene Order and Chromosome Dynamics Coordinate Spatiotemporal Gene Expression During the Bacterial Growth Cycle

Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle

Patrick Sobetzkoa, Andrew Traversb,c, and Georgi Muskhelishvilia,1

aSchool of Engineering and Science, Jacobs University Bremen, D-28759 Bremen, Germany; bMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and cFondation Pierre-Gilles de Gennes pour la Recherche, Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, 94235 Cachan, France

Edited by Sankar Adhya, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved November 23, 2011 (received for review May 23, 2011) In Escherichia coli crosstalk between DNA supercoiling, nucleoid-as- selection of mutations in fis and tRNA dihydrouridine synthase sociated proteins and major RNA polymerase σ initiation factors (dusB) (essential for fis expression) and also in topA (31), as well as regulates growth phase-dependent gene transcription. We show in rpoC (the β′ subunit of RNAP) under conditions of adaptive that the highly conserved spatial ordering of relevant genes along evolution (32). the chromosomal replichores largely corresponds both to their tem- Although there is substantial evidence for integrated regulation poral expression patterns during growth and to an inferred gradient of NAPs, DNA superhelicity, and RNAP selectivity during the of DNA superhelical density from the origin to the terminus. Genes growth cycle, the mechanism by which this regulation is accom- implicated in similar functions are related mainly in trans across the plished remains obscure. We report here that the conserved or- chromosomal replichores, whereas DNA-binding transcriptional reg- dering of the stage-specific regulatory genes and their targets along ulators interact predominantly with targets in cis along the repli- the replichores corresponds with their temporal expression pat- chores. We also demonstrate that macrodomains (the individual terns during the growth cycle. We propose that this ordering structural partitions of the chromosome) are regulated differently. reflects a gradient of DNA gyrase-binding sites and hence negative We infer that spatial and temporal variation of DNA superhelicity superhelicity from chromosomal origin (OriC) to terminus (Ter) during the growth cycle coordinates oxygen and nutrient availabil- of replication and that the generation of this superhelicity gradient ity with global chromosome structure, thus providing a mechanistic is coupled to energy availability. During the growth cycle changes insight into how the organization of a complete bacterial chromo- in local superhelicity drive morphological changes in chromosome some encodes a spatiotemporal program integrating DNA replica- structure that facilitate the integration of DNA replication and tion and global gene expression. gene expression. gene order conservation | transcriptional regulatory network | Results protein gradients Gene Order. We observed that the ordering, relative to OriC, of the genes encoding the NAPs specific to particular stages of the n Escherichia coli cells the physiological transitions induced by growth cycle [i.e., fis, hupA, β subunit of histone-like protein from Ithe changing growth environment are accompanied by changes E. coli strain U93 (hupB), suppression of td phenotype (stpA), lrp, in DNA superhelical density (1–3), nucleoid structure (4–6), and dps, cbpA, and β subunit of integration host factor (ihfB)] ap- the promoter selectivity of the RNA polymerase (RNAP) holo- proximately reflects their relative abundance during the growth enzyme (3, 7). During the growth cycle both the relative and cycle (8, 9). With one exception, hns, the NAPs associated with absolute concentrations of the abundant nucleoid-associated the higher overall superhelicity characteristic of exponential proteins (NAPs; Table S1) change substantially and correspond- growth are closer to the origin. Conversely, those associated with ingly generate bacterial chromatin of variable composition (8, 9). the lower superhelical density characteristic of the stationary The NAPs stabilize distinct supercoil structures (10–12) selec- phase are closer to the replication terminus (Fig. 1B). The or- tively favoring particular RNAP holoenzymes (13–15). These dering of the genes for the transcriptional-machinery components variable nucleoprotein complexes modulate DNA topology during exhibits a pattern similar to that of the NAPs, with rpoD, encoding the growth cycle (Fig. 1A), optimizing the channeling of supercoil the σ70 factor for vegetative growth, located closer to OriC than energy into appropriate metabolic pathways (16, 17). rpoS, encoding the stationary phase σS factor. Similarly, gyrB (but The expression of the genes determining superhelical density, not gyrA), encoding subunit B of DNA gyrase, is located in close polymerase selectivity, and nucleoid structure is coordinated by proximity to OriC. Gyrase increases negative superhelicity, es- cross-regulation. Thus, factor for inversion stimulation (FIS), pecially with the higher ATP/ADP ratios prevailing on nutritional a NAP abundant during the early exponential phase (18), regulates shift-up (33). In contrast, both topA and topoisomerase III (topB), expression not only of the superhelicity determinants DNA gyrase encoding the DNA-relaxing topoisomerases, are closer to the subunits A and B (gyrA and gyrB) and topoisomerase I (topA)(19– 21) but also other NAP-encoding genes including hns, α subunit of histone-like protein from E. coli strain U93 (hupA), and DNA Author contributions: A.T. and G.M. designed research; P.S. performed research; P.S., A.T., binding protein from starved cells (dps)(22–24) and components and G.M. analyzed data; and P.S., A.T., and G.M. wrote the paper. 38 of the transcription machinery such as σ subunit of RNA poly- The authors declare no conflict of interest. merase rpoS (25). Similarly mutations affecting the selectivity of This article is a PNAS Direct Submission. fl – RNAP in uence NAP production (26 28). Again, mutations in the Freely available online through the PNAS open access option. genes controlling DNA superhelicity affect the production of both 1To whom correspondence should be addressed. E-mail: g.muskhelishvili@jacobs-university. NAPs and the basal transcription machinery (27, 29). This pattern de. of integrated control constitutes a heterarchical network co- See Author Summary on page 355. ordinating chromosome structure with cellular metabolism (27, 28, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 30). A further pointer to this integrated network is the observed 1073/pnas.1108229109/-/DCSupplemental.

E42–E50 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Downloaded by guest on September 27, 2021 A PNAS PLUS

NAP abundance

RNA polymerase sigma factors S

Plasmid superhelical density ( ) -0.068 -0.043

Growth stage Shift up Early log Mid/late log Transition Stationary (ppGpp spike) B Chromosomal Right Ori NS Right Ter macrodomains OriC Ter Left Ori NS Left Ter Aerobic/anaerobic metabolism, BAC E arcA H seqA rmf fnr DNA replication, OriC Ter rrn genes, etc atp arcB G dnaA D yacG topA DNA topology OriC Ter gyrB parC gyrA sbmC topB parE dps ihfB hupA hfq hupB lrp cbpA hns NAPs OriC Ter crp fis stpA ihfA rsd nusG rpoBC crl rho RNA polymerase fecI dksA OriC Ter modulators rpoZ greB rpoD ssrS rpoS rpoE fliA

rpoH GENETICS rpoA nusA rpoN greA

Fig. 1. Spatiotemporal organization of chromosomal expression. (A) Temporal changes of NAPs, RNAP composition, and average plasmid DNA superhelicity (σ) during bacterial growth. The phases of growth cycle are correlated with preferred expression of particular NAPs, RNAP holoenzymes and the supercoiling temporal gradient are indicated below. A transient increase in guanosine tetraphosphate (ppGpp) levels occurs at the transition between the exponential and stationary phases. (B) Spatial ordering of regulatory genes on the E. coli chromosome along the OriC–Ter axis. (Top line) Correspondence of macrodomains deﬁned by Valens et al. (40) to linear map. (First bar) Selected genes involved in aerobic/anaerobic metabolism (dark blue), DNA replication (orange), rrn genes (red), and transition phase (brown). Genes on the clockwise (right) replichore are above the bar, and genes on anti-clockwise (left) replichore are below the bar. The atp operon encodes ATP synthase. arcA/arcB encode a two-component system active under microaerobic conditions (61, 62). ArcA also represses rpoS (63). fnr has a dominant role under more strictly anaerobic conditions (61). dnaA, encoding the principal initiator of DNA replication, maps close to OriC, whereas seqA, aninhibitorofreplication initiation at OriC, maps closer to Ter. rmf decreases the availability of ribosomes and maps to a macrodomain immediately adjacent to the Ter macrodomain. (Second bar) Selected genes involved in control of DNA topology. gyrB, a component of DNA gyrase responsible for increasing negative superhelicity, maps close to OriC, whereas the gyrase inhibitor susceptibility to B17 microcin, locus C (sbmC), and topA and topB, both responsible for relaxing DNA, map either close to or within the Ter macrodomain. DNA gyrase inhibitor (yacG), encoding an inhibitor of GyrB, maps close to the center. Chromosomal partition genes C and E (parC and parE) encode the subunits of topoisomerase IV, responsible for decatenation of newly replicated DNA in the terminal region (64) and relaxation of negative supercoils (65). (Third bar) Selected genes encoding NAPs. The NAP-encoding gene closest to OriC is hupA, encoding histone-like protein from E. coli strain U93 (HU)α. Its early

expression relative to hupB, encoding Huβ (9), could buffer high negative superhelicity generated by DNA gyrase (36). HUα2 and HUαβ, but not HUβ2,constrainhigh superhelical densities in vitro (9). A mutation in hupA both increases growth rate and antagonizes histone-like nucleoid-structuring protein (H-NS) regulation of certain transcription units (6). High frequency of recombination (Hfq) is a nucleic acid-binding protein whose major role is that of an RNA chaperone, but it also may act as a DNA-binding NAP (8). lrp is activated by ppGpp (38). (Fourth bar) Selected genes involved in modulating RNAP activity, including σ factor-utilization regulators, secondary channel-binding proteins, termination/elongation factors, and RNAP subunits. σ factor-utilization regulators (light green): ω subunit of RNA polymerase (rpoZ), mapping close to the origin, encodes the ω subunit of RNAP, which confers a preference for utilization of σ70 (28). Regulator of sigma D (rsd) encodes an anti-σ70 (66), whereas crl confers a strong preference for σS utilization (67). Note that both rpoZ and crl mapclosertoOriCthandotherespectiveσ factors whose activity they affect. The encoded regulatory pattern thus reﬂects a shift from predominantly σ70 use close to OriC to σS availability in the central region of the chromosome. Secondary channel-binding proteins (plum): growth regulator A and B, transcription elongation factors (greA and greB) both map in the region containing many genes expressed during rapid growth. GreA has been shown to stimulate initiation and transcription of genes involved in aerobic metabolism, including the atp operon (68, 69) as well as the rrnB P1 promoter in vitro (70, but also see ref. 71). DksA, like the plasmid-encoded quorum sensing regulator (TraR) protein (72), inhibits ribosomal protein promoters and rrn initiation (73, 74) and is more distant from OriC than is greA. In vivo it would act to reduce the rate of rrn initiation and hence antagonize transcription foci formation. Termination/elongation factors (red): The termination factor Rho is encoded by a gene located very close to OriC. This location may compensate for the antagonistic effect of high negative superhelicity on transcription termination, which involves the rewinding of DNA. RNAP subunits: The map positions relative to OriC of rpoD and rpoS, respectively encoding σ70 and σS, correspond to their relative order of temporal expression.

Ter. Spatial organization of genes modifying the transcription (dksA), curly (crl)] and genes sustaining catabolism and energy machinery [transcriptional terminator Rho (rho), the N utiliza- production under aerobic [ATP synthase (atp) operon], micro- tion substance gene (nus) factors, the gre factors, dnaK suppressor aerobic [two component signal transduction system (arcA/B)], and

Sobetzko et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | E43 Downloaded by guest on September 27, 2021 A such genes from the origin and terminus was highly conserved (Fig. 2A). Similar analysis of all orthologous genes demonstrated that, although the relative distance was conserved, the speciﬁc replichore was not conserved to the same extent (Fig. 2B). Simulations supported this observed bias (Fig. S1). Furthermore, essentially the same picture emerged when we analyzed the more distantly related Gram-positive bacteria (Fig. S2 A and B). We conclude that conservation of relative distance along the OriC–Ter axis overrides the replichore coherence.

Targets. Not only are the genes coordinating the major regulatory pathways ordered; their targets are ordered as well. Analyses of the distribution density of binding sites for Eσ70 and EσS holoenzymes compiled in RegulonDB (34) show opposite spatial biases. For the vegetative σ70 factor the highest percentage of targets is found around the origin, whereas for the stationary phase σS factor the highest target density is close to the terminus (Fig. 3 A and B), consistent with both the closer location of rpoD to OriC and the temporal division of labor between σ70 and σS during the bacterial growth cycle (7). Similarly, the average density of binding sites for DNA gyrase (35) diminishes by five- to 10-fold (Fig. 3C) from OriC to Ter (36). This organization could generate a gradient of superhelical density (Fig. 3D) correlating with that of Eσ70 targets and anti-correlating with EσS targets, as expected from the B opposite supercoiling preferences of these holoenzymes (3, 28) and in keeping with the requirement of high negative superhelicity for initiation of OriC replication (37). For the major NAPs, despite distinct chromosomal locations and abundances during the growth cycle, a high percentage of the binding sites compiled in RegulonDB occurs around the origin (Fig. 3 E–H). Among the NAPs encoded in the Ter- proximal region, only integration host factor (IHF) targets activating binding sites in the vicinity of Ter (Fig. 3F), whereas the stationary-phase regulator leucine responsive protein (LRP) (38) and the global repressor H-NS (39) both preferentially target the OriC-proximal region (Fig. 3 G and H). Additionally HU, the major supercoil-constraining NAP for which no binding site information is available, has distinct and opposite functional effects at the OriC and Ter ends of the chromosome, respectively reducing and increasing transcription (Fig. S3). To explore the relevance of this apparent chromosomal ordering Fig. 2. Arrangement of important regulatory elements according to their po- of regulators for chromosomal expression, we used the Gene On- γ sition relative to OriC in the -Proteobacteria. (A) Genes located on the right and tology (GO) database describing the gene products in terms of as- left replichores are indicated above and below the chart, respectively, as in Fig.1B. Horizontal bars show spatial distributions of orthologs; black color indi- sociated metabolic processes and investigated the spatial organi- cates the highest density for each individual distribution. The plot shows a strong zation of functional groups of genes. Many of these genes would be conservation of chromosomal positions suggesting that origin-focused gene theultimatetargetsoftheregulation. Using scanning windows of positioning is a major selection criterion in γ-Proteobacteria. (B) Relationship variable sizes (0.1–0.5 Mb), we mapped the genes in the GO data- among the phylogenetic distance, the correlation of distance to origin, and the base, identifying the significant matches between functionally com- replichore coherence (the conserved replichore identity of orthologs) for all plementing windows on the chromosome (Figs. S4 and S5). We γ-Proteobacteria. The points represent the data on pair-wise species compar- determined the ratio of all significant combinations for cis (matching isons computed using the Pearson correlation coefficient of either distances to windows located on the same replichore) and trans (matching win- origin or replichore identity (right/left) of all orthologous pairs. The points are dows on distinct replichores) arrangements. Although the GO tree color-coded in the 3D plot. Red indicates a higher correlation of distance to organization comprises 13 complexity levels from the most broad origin than replichore coherence; blue indicates higher replichore coherence. fi fi The predominance of red points indicates the stronger conservation of distance down to speci c functions, most signi cant matches were observed to origin. The phylogenetic distances in B were derived from the tree of γ-Pro- at levels three and four (Figs. S6 and S7). Fig. 4A shows that func- teobacteria (http://www.cbrg.ethz.ch/research/orthologous/speciestrees). tionally matching groups are localized predominantly on opposite replichores within the rrn macrodomain (36) comprising OriC and the nonstructured left (LNS) and right (RNS) macrodomains (40). anaerobic (fnr) conditions, as well as those involved in activation Importantly, the predominantly trans organization of these groups and negative modulation of replication [DNA replication initiator was significant only when the chromosomal gene order was aligned protein DnaA (dnaA) and sequestration of origin (seqA)] and along the OriC–Ter axis. We infer that a majority of the functionally translation [ribosomal RNA (rrn) operons and ribosome matura- related groups are organized at comparable distances from the tion factor (rmf)], exhibit a similar pattern of chromosomal or- center of symmetry at OriC and are related in trans across the dering (legend of Fig. 1B and Table S2). replichores. We denote these spatially coordinated matching GO We asked whether the gene order of regulatory elements as- groups of genes as “maGOGs.” sociated with the temporal pattern of transcription in Escherichia We next analyzed the interactions between the E. coli DNA- coli was conserved in other γ-Proteobacteria. Analysis of 131 binding transcriptional regulators and their targets compiled in γ-proteobacterial genomes showed that the relative distance of the RegulonDB in the form of a static transcriptional regulatory

E44 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al. Downloaded by guest on September 27, 2021 AE PNAS PLUS

D H GENETICS

Fig. 3. Organization of binding sites for the major σ initiation factors, DNA, gyrase, and NAPs in the chromosome (RegulonDB). Distributions were calculated by using a sliding window of 400 kb and normalizing over the total gene number for each window. The replichores are organized from OriC to Ter (left to right). The frequency distributions (ordinate) are plotted above the zero in the ordinate for the right replichore and below the zero for the left replichore. (A) Genomic distributions for RNAPσ70-regulated promoters. (B) Genomic distributions for σS-regulated promoters. (C) Genomic distributions of gyrase-binding sites. (D) Inferred gradient of negative superhelical density along the OriC–Ter axis in exponentially growing cells. (E) Genomic distribution of FIS-binding sites. (F) Genomic distribution of IHF-binding sites. (G) Genomic distribution of H-NS–binding sites. (H) Genomic distribution of LRP-binding sites. The chromosomal position for each regulator gene is indicated, and the direction of the effect is rainbow color-coded with blue for repression and red for activation. The Ori, Ter, left, and right macrodomains (green, cyan, blue, and red lines, respectively) are indicated on the chromosomal replichores above and below the distributions.

network (TRN). These analyses again revealed macrodomains in analyses shown in Figs. 3 and 4 use the static E. coli Regulon which spatially coordinated interactions between the regulators database, which contains neither temporal nor spatial ordering and targets were significantly enriched. However, in contrast to information. To analyze the dynamic organization of communi- the spatial organization of maGOGs, significant TRN inter- cations in the effective transcript profiles (41) obtained under actions occur mainly in cis along the replichores, largely corre- defined conditions, we integrated the maGOGs, TRN, and sponding to intermacrodomain communication (Fig. 4B). couplon networks in a single combined heterarchical network We also analyzed the spatial organization of couplons, entities (HEN). In such a network, as opposed to a hierarchical network, corresponding to intersections of regulons of distinct σ factors there is no subordination to one single dominant component. and NAPs and containing functionally related genes (27). Cou- The HEN (Fig. 4D) served as a template for mapping the ef- plon analyses again demonstrated preferential communications fective transcript profiles. The observed gene ordering predicted in trans across the replichores (Fig. 4C). We infer that chromo- that relative transcription during exponential and stationary somal arrangement of functionally related genes and the spatial growth phases would be ordered in a similar spatial manner communications between transcriptional regulators and their relative to OriC and Ter. The results fully confirmed this pre- targets are essentially orthogonal with respect to each other. diction, clearly demonstrating a division of labor between the OriC and Ter chromosomal ends in temporal expression profiles. Expression Profiles. Any valid regulatory network must describe In exponentially growing wild-type cells the OriC-proximal region meaningfully the changes in gene expression both during normal was transcriptionally more active than the Ter-proximal region growth and when expression is perturbed by mutation. The with the active regions corresponding largely to the rrn domain

Sobetzko et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | E45 Downloaded by guest on September 27, 2021 A B

C D

Fig. 4. Organization of functional groups and regulatory communications in the E. coli chromosome. (A) maGOGs. (B) TRN. (C) Couplons. (D) HEN. The circular genome is represented as a pair of multicolored parallel lines corresponding to the right (Upper line) and left (Lower line) replichores. On the replichores the macrodomains (colored as in Fig. 3) and the rrn functional domain (orange dashed line) are indicated. All trans communications occur between the upper and lower lines, whereas cis communications occur along the lines. The order of regulatory genes on the right and left replichores is indicated above and below each network, respectively, organized from OriC to Ter.

comprising the Ori and LNS macrodomains (Fig. 5 A and B). and F). Importantly, although the effects of σ factors mainly in- Similar spatiotemporal division of labor was observed in an rpoS volved trans communications, consistent with the orthogonal or- mutant lacking σS, but the pattern relative to wild-type cells was ganization of regulatory communications in HEN, the mutations reversed, with the Ter-proximal region being more favored during of ﬁs and hns genes also substantially affected the cis communi- the exponential phase and the OriC-proximal region during the cations, which were largely buffered in wild-type cells (Fig. 5 G– stationary phase (Fig. 5 C and D). Also, in an rpoZ mutant fa- L). Furthermore, some of these spatiotemporal patterns could be voring the EσS holoenzyme (28), the Ter-proximal region was closely imitated by manipulating the composition of NAPs and favored during the exponential phase, an effect that could be re- DNA superhelicity (Fig. S8), validating the usefulness of HEN for versed by overproduction of σ70 activating the OriC end (Fig. 5 E gene-expression analyses.

E46 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al. Downloaded by guest on September 27, 2021 Discussion PNAS PLUS A C E We have demonstrated that during the E. coli growth cycle in batch culture the temporal pattern of stage-specific gene expression corresponds largely with the gene order along the two replichores. This correspondence is apparent not only for the principal regulatory genes but also for their targets. Importantly, this property is a highly conserved characteristic of the replichores in both Gram-negative γ-Proteobacteria and Gram-posi- B D F tive bacteria, implying that the ordering is related to DNA replication. Moreover, the most conserved property for a particular gene appears to be not the precise location but the relative distance of the gene from OriC and Ter, independent of replichore (Fig. 2 and Figs. S1 and S2 A and B). This finding suggests that the selective determinant is itself a variable property that G I K also is dependent on the relative OriC/Ter distance and is consistent with macrodomains sharing many functions (Fig. S9). In fast-growing E. coli the bidirectional replication starting from OriC creates a gradient of gene dosage from OriC to Ter, such that the resulting origin-proximal relative increase in gene dosage potentially could increase gene expression in that region. Indeed, it is precisely this region that is preferentially active during exponential growth (Fig. 5A). However, variation in gene dosage is likely to be H J L only one of a number of regulatory influences. Thus, the origin- proximal region contains a five- to 10-fold higher average density of gyrase-binding sites than the Ter-proximal region (Fig. 3C)and also is the preferred target region for Eσ70 holoenzyme and for FIS protein (Fig. 3 A and E), implicated in evolutionary modulation of DNA superhelicity (25). We infer that the gradient of gyrase M N binding sites is indicative of potential gradients of negative superhelical density from OriC to Ter along the replichores (Fig. 3D). The existence of such gradients corresponds well with both the pattern of holoenzyme and NAP targets and the greater density of supercoiling-sensitive genes (e.g., rpoZ, fis, hupA,andrrn)in Fig. 5. Spatiotemporal HEN patterns. The circular genome is represented as the OriC end (16, 36, 42), as well as with the activation of the rrn GENETICS a pair of thin parallel lines corresponding to the right (Upper line) and left macrodomain under conditions of high negative superhelicity (Lower line) replichores as in Fig. 4. Macrodomains are indicated in color on the during exponential growth (Fig. S10 A and B). The gradient of right (Upper) and left (Lower) replichores organized from OriC to Ter. (A)Ef- gyrase-binding sites would provide a simple mechanism for cou- fective HEN (eHEN) pattern of exponentially growing wild-type cells. (B)eHEN pling energy availability to superhelical density (2, 33) and, be- pattern of transcripts from stationary wild-type cells. Red indicates coherently cause initiation of DNA replication is enhanced by high negative enhanced and blue indicates coherently reduced numbers of expressed genes superhelicity (37), also to replication itself. in matching windows; black indicates an absence of coherence. Note the fi switch in the activation of the OriC and Ter ends of the chromosome at the Although the spatial order of selected stage-speci c genes transition from exponential to stationary growth. (C) eHEN pattern of rpoS corresponds well to the temporal order of expression during the cells lacking σS obtained during exponential phase. (D) eHEN pattern of rpoS growth cycle, the position of certain other genes, although highly cells in stationary phase. Note the spatiotemporal inversion of the activation of conserved, does not. Genes in this category in E. coli include hns the OriC and Ter ends of the chromosome with respect to wild-type cells. (E) and gyrA. Although in Gram-positive bacteria gyrA and gyrB map σS eHEN pattern of exponentially growing rpoZ cells favoring E .(F)AsinE, but in close proximity to OriC (Fig. S2C), a rationale for the separate σ70 σ70 the cells were overproducing from an episome (28). Note the -de- conserved locations of gyrA and gyrB in γ-Proteobacteria is not pendent coherent activation and repression of communications in the OriC and Ter ends of the chromosome, respectively. (G and H) eHEN patterns of obvious. On shift-up expression of gyrB increases to a propor- wild-type cells grown under conditions of high and low superhelicity, re- tionally much greater extent than expression of gyrA (19). GyrA spectively. (I and J) Patterns of hns-mutant cells grown under conditions of and GyrB together form a heterotetramer, and if GyrB were high and low superhelicity, respectively. Note the supercoiling-dependent limiting the distinct locations would favor the use of the gyrase- coherent activation and repression of communications in the left and right binding sites in the origin-proximal region. We hope to clarify any replichores, respectively. (K and L) eHEN patterns of fis-mutant cells grown requirement for separate placement of the gyrA and gyrB genes in under conditions of high and low superhelicity, respectively. Note the coherent E. coli by switching their chromosomal positions. activation of distinct supercoiling-dependent cis and trans communications. In Both FIS and H-NS principally target the origin-proximal re- A–D the cells were grown in minimal medium. In E–L the cells were harvested gion, potentially delimiting short topological domains (43). How- during exponential growth in rich double-YT medium. (M and N) Model of fi chromosomal morphology changing with DNA superhelicity and NAP gra- ever, s is expressed at maximal levels during the exponential dients on transition from exponential (M) to stationary (N) growth. For clarity, phase and maps in the origin-proximal region, in contrast to hns, only the interactions between the gradients of FIS (pink) and H-NS (blue) are which maps in the Ter macrodomain. In this context Montero shown. Although FIS levels decline dramatically on transition to stationary Llopis et al. (44) recently reported that both Escherichia coli and phase, the compaction of the nucleoid along the OriC–Ter axis enables H-NS to Caulobacter crescentus spatially organize translation so that the establish repression. The chromosome is depicted as a plectoneme, but the mRNA product of a gene is translated in close proximity to its model is equally consistent with a toroidal scaffold, which would maintain the position in the nucleoid. This observation has profound implica- separation of the replichores (49, 50). The macrodomains are indicated by colors, and approximate chromosomal positions of the fis and hns genes are tions for gene regulation and chromosome structure. If NAP shown. The expression data for mapping onto the HEN connectivity patterns production is localized, then diffusion of the resultant DNA- were taken from Dong and Schellhorn (59) in A–D, from Geertz et al. (28) in E binding proteins within the nucleoid will be anomalous, generating and F, and from Blot et al. (16) in G–L. concentration gradients. The outcome for nucleoid organization at

Sobetzko et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | E47 Downloaded by guest on September 27, 2021 a given locus then depends on the relative local concentrations of active in replication initiation (53) and coordinating RNAP ac- different NAPs, which in turn depend on the distance from the site tivity with energy availability (54). We propose that gyrase ac- of production and the total number of molecules available. In this tivity creates a gradient of superhelical density corresponding to model FIS and H-NS would be expected to be more dominant on the gradient of gyrase-binding sites (Fig. 3 C and D and Fig. S10 nucleoid structure and function in the origin-proximal region A and B) such that the selectively increased superhelicity of the during the exponential and stationary phases, respectively. origin-proximal region and the resultant change in chromosome The relation of NAP function to negative superhelicity is con- morphology precede the initiation of replication. Such selectivity sistent with the concept that NAPs can act to buffer superhelical suggests a lower superhelical density in the Ter region, consistent density not only at the local level, as previously reported for the with the finding that in the prereplicative state in slow-growing rrnA P1 promoter (45), but also at the more global level of the cells the Ter region is extended (55). Concomitantly the in- – chromosome and macrodomains (Fig. 5 G L). In this respect creased spatial separation between origin-proximal H-NS target a correlation between the gradient of gyrase-binding sites and the genes and the Ter-proximal location of the hns gene (Fig. 5M) functional effect of HU (36) is particularly striking (Fig. S3). In the would attenuate the silencing effect H-NS (39, 56, 57) on the rrn context of the transcriptional effect of HU, the apparently macrodomain. On transition to stationary phase. a dramatic anomalous positions of the rrnG and rrnH genes approximately decline of FIS levels (18) in conjunction with DNA relaxation halfway between OriC and Ter delimit the rrn functional macro- (Fig. S10 A and B) and compaction of the nucleoid would enable domain (36). Importantly, although all seven rRNA operons have fi H-NS, as well as the stationary-phase regulator LRP (38), to been shown to be necessary for rapid adaptation, only ve are reestablish efficient repression (Fig. 5N) in the vicinity of repli- necessary to support near-optimal growth (46), and it is not known cation origin. This model provides a dynamic mechanism for the how the rebalancing of rRNA transcription after shift-up is dis- concerted impact of the NAPs and DNA superhelicity on chro- tributed among the rrn operons. Also, in a strain lacking ppGpp mosome structure and cellular physiology (4–6, 9, 10, 16, 17, 19, (the negative regulator of rRNA transcription), the regions con- 36, 39, 58). taining up-regulated genes whose expression is stimulated by high negative superhelicity are in close proximity to rrnG and rrnH (Fig. Conclusion S10 C and D). This finding together with the propensity for forming The conservation of gene order and spatial organization of variable transcription foci (47) raises the possibility that, in growing regulator–target interactions in chromosomal macrodomains cells with a full complement of rrn operons, the regulation of in- fi dividual rrn operons depends on their position in the chromosome. reported in this paper argue that the gene order speci es the A related issue is how the scattered distribution of tRNA temporal pattern of gene expression during the bacterial growth genes around the chromosome can be reconciled with chromo- cycle. Our work thus illuminates why the timing of the expression somal positioning (Fig. S11). Unlike rRNA genes, the expression of a gene is linked to its position on the chromosome, providing of tRNA genes must be consistent both with growth and with a view of the bacterial nucleoid as a highly organized dynamic differences in the relative amounts of iso-accepting tRNAs and entity optimized for coordinating oxygen and nutrient availability codon use at different growth rates (ref. 48 and legend of Fig. with spatiotemporal gene expression during rapid growth and S11). For both rRNA and iso-accepting tRNA genes, as for other its cessation. genes described here, distance from the origin is more conserved Materials and Methods than replichore (Fig. S11B). Identification of Functional Groups in the GO Tree and Modeling of the TFGs. To Topological Model for Temporal Gene Expression. The deduced logic address the similarity of chromosomal regions, we applied a sliding-window of spatial communications in the E. coli chromosome suggests approach using a 500-kb window size and a 4-kb window shift creating 1,160 windows, each containing about 500 genes. All genes within a window were a simple topological model of the circular chromosome folded as assigned to their specific locations on the GO tree (http://www.geneontology. a negative supercoil in which close spatial proximity of replichores fi fl org/). For a xed level of the GO tree we summed up all genes assigned to either within the rrn macrodomain comprising the Ori and ank- the specific subtree, determining a unique one-dimensional pattern of GO- fl ing exible macrodomains or between the rrn and Ter macro- subtree (cluster) sizes for each window. Subsequently the similarity score of domains supports alternative communication during exponential two windows (i and j) was determined by the equation: and stationary growth. These morphological transitions could occur as a consequence of changes in superhelical density, which c ∑ ni;k ∗nj;k fi k¼1 would affect the con guration of DNA directly and also would s ; ¼ fi i j c c alter the relative af nities of the different NAPs for DNA. The ∑ ni;k ∗ ∑ nj;k alternative configurations, with varying OriC and Ter separation, k¼1 k¼1 are consistent with the formation of transcription foci (47), the where c is the number of clusters on the current GO-tree level, and n is the reorganization and physical extension of nucleoids during expo- number of genes in a certain branch of this level, reflecting their covariance – nential growth (49 51), and the compaction of nucleoids in the with a mean that equals zero. Hence, high similarity scores (s values) indicate stationary phase (51, 52). This last effect is counteracted by the a high correlation of the functional composition. All s values were compared activator of rRNA operons FIS, which is abundant in exponential with scores of 10,000 random genomes, generated by a random remapping phase (4, 18), suggesting that the conformational transition in the of gene (operon) IDs to gene (operon) positions. For subsequent analyses nucleoid is associated with changing gradients of competing NAPs window pairs with a P value < 0.05 were considered significant, and all (Fig. 5 M and N). Although the extent to which gene expression others, including scores of overlapping windows [distance(i,j) <500 kb], were has a linear relationship with gene copy number is unknown, it is excluded. However, higher significance values up to a false-discovery rate of conceivable that the relative increase in gene dosage resulting 0.05 indicated no qualitative change. In practice, the exclusion of over- from the initiation of replication would increase the competitive lapping windows introduces a bias toward trans matches. To address this advantage of regulators encoded in the origin-proximal region, issue and to compile a reference data set with the same bias, we shifted the origin position along the chromosome and compared cis/trans ratios with whereas a balanced OriC-to-Ter stoichiometry on cessation of the native origin location. Subsequently, the significance of the cis/trans growth would abolish such an advantage (Fig. S12). ratio peaks for a shifted origin position was compared with 10,000 random How could such morphological transitions be coupled to en- gene sets with the same number of best-scoring matches to rule out random ergy availability? A primary response to nutritional shift-up un- noise peaks. GO levels three and four turned out to be statistically signifi- der aerobic conditions is an increase in the ATP/ADP ratio (2, cant; lower GO levels lacked a reasonable functional resolution, whereas 33) that activates DNA gyrase, favoring the DnaA–ATP complex higher levels represented an extremely detailed functional resolution that

E48 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al. Downloaded by guest on September 27, 2021 suffered from varying resolutions among distinct functional clusters because Expression Patterns. The expression data for mapping onto the HEN con- PNAS PLUS of a nonuniform depth of the GO tree. nectivity patterns were taken from Dong and Schellhorn (59) in Fig. 5 A–D, from Geertz et al. (28) in Fig. 5 E and F, and from Blot et al. (16) in Fig. 5 G–L. Modeling of the TRN. In correspondence with the GO-tree function analysis, we parsed the TRN communication along the chromosome by counting the Phylogenetic Analysis. In the phylum of γ-Proteobacteria the majority of reg- number of connections between the subnetworks of any two 500-kb win- ulators is conserved in a plethora of species including human and plant dows normalized by the total number of connections spreading from both pathogens such as Vibrio cholerae and Pseudomonas syringae.Intotalwein- windows into the genome. Using procedures analogous to GO similarity vestigated 131 species (using a reciprocal best blast hit approach at the protein analysis, we determined highly connected regions on the chromosome and determined the peaks for the cis/trans ratio by shifting the origin. sequence level followed by the determination of orthologous clusters by amodiﬁed Girvan–Newman algorithm, resulting in densely connected clusters ≥ Couplon Similarity. As a measure of couplon similarity, we determined for (node degree n/2), where n denotes the cluster size. Species information each couplon (27) whether the number of contained genes for the current containing protein sequences was derived from the RefSeq sequence database window was greater or less than the expected number of genes. Hence, the and origin positions using the DoriC database (60). The phylogenetic distances similarity of two windows equals the number of couplons coherently over- in Fig. 2B were derived from the tree of γ-Proteobacteria (http://www.cbrg. ﬁ or underrepresented in both windows. The determination of the signi - ethz.ch/research/orthologous/speciestrees). The same methodology was ap- cance of matches and cis/trans ratio peaks was carried out as for the GO-tree plied for analysis of all the annotated genomes of Gram-positive bacteria. similarity with random sets of both genes and operons. ACKNOWLEDGMENTS. The authors thank Malcolm Buckle and Sylvie Rimsky for Regulator Targets. To determine target site frequency distributions, the rel- fruitful discussions. This work was supported by grants from the Ecole Normale evant data derived from the RegulonDB database were analyzed using Supérieure de Cachan and Deutsche Forschungsgemeinschaft (to G.M.). A.T. re- a sliding 400-kb window with the map position at the window midpoint. ceived a Chaire d’Excellence award from l’Agence Nationale de la Recherche.

1. Balke VL, Gralla JD (1987) Changes in the linking number of supercoiled DNA 24. Grainger DC, Goldberg MD, Lee DJ, Busby SJ (2008) Selective repression by Fis and H- accompany growth transitions in Escherichia coli. J Bacteriol 169:4499–4506. NS at the Escherichia coli dps promoter. Mol Microbiol 68:1366–1377. 2. Hsieh LS, Burger RM, Drlica K (1991) Bacterial DNA supercoiling and [ATP]/[ADP]. 25. Hirsch M, Elliott T (2005) Fis regulates transcriptional induction of RpoS in Salmonella Changes associated with a transition to anaerobic growth. J Mol Biol 219:443–450. enterica. J Bacteriol 187:1568–1580. 3. Bordes P, et al. (2003) DNA supercoiling contributes to disconnect sigmaS 26. Zhou YN, Jin DJ (1998) The rpoB mutants destabilizing initiation complexes at accumulation from sigmaS-dependent transcription in Escherichia coli. Mol Microbiol stringently controlled promoters behave like “stringent” RNA polymerases in 48:561–571. Escherichia coli. Proc Natl Acad Sci USA 95:2908–2913. 4. Ohniwa RL, et al. (2006) Dynamic state of DNA topology is essential for genome 27. Muskhelishvili G, Sobetzko P, Geertz M, Berger M (2010) General organisational condensation in bacteria. EMBO J 25:5591–5602. principles of the transcriptional regulation system: A tree or a circle? Mol Biosyst 6: 5. Rimsky S, Travers A (2011) Pervasive regulation of nucleoid structure and function by 662–676. nucleoid-associated proteins. Curr Opin Microbiol 14:136–141. 28. Geertz M, et al. (2011) Structural coupling between RNA polymerase composition and 6. Kar S, Edgar R, Adhya S (2005) Nucleoid remodeling by an altered HU protein: DNA supercoiling in coordinating transcription: A global role for the omega subunit? Reorganization of the transcription program. Proc Natl Acad Sci USA 102: mBio 2, 4; e00034-11. – 16397 16402. 29. Peter BJ, et al. (2004) Genomic transcriptional response to loss of chromosomal 7. Ishihama A (2000) Functional modulation of Escherichia coli RNA polymerase. Annu supercoiling in Escherichia coli. Genome Biol 5:R87. Rev Microbiol 54:499–518. 30. Travers A, Muskhelishvili G (2005) DNA supercoiling - a global transcriptional GENETICS 8. Ali Azam T, Iwata A, Nishimura A, Ueda S, Ishihama A (1999) Growth phase- regulator for enterobacterial growth? Nat Rev Microbiol 3:157–169. dependent variation in protein composition of the Escherichia coli nucleoid. 31. Crozat E, et al. (2010) Parallel genetic and phenotypic evolution of DNA superhelicity – J Bacteriol 181:6361 6370. in experimental populations of Escherichia coli. Mol Biol Evol 27:2113–2128. 9. Claret L, Rouvière-Yaniv J (1997) Variation in HU composition during growth of 32. Conrad TM, et al. (2010) RNA polymerase mutants found through adaptive evolution Escherichia coli: The heterodimer is required for long term survival. J Mol Biol 273: reprogram Escherichia coli for optimal growth in minimal media. Proc Natl Acad Sci 93–104. USA 107:20500–20505. 10. Dame RT (2005) The role of nucleoid-associated proteins in the organization and 33. van Workum M, et al. (1996) DNA supercoiling depends on the phosphorylation compaction of bacterial chromatin. Mol Microbiol 56:858–870. potential in Escherichia coli. Mol Microbiol 20:351–360. 11. Guo F, Adhya S (2007) Spiral structure of Escherichia coli HUalphabeta provides 34. Gama-Castro S, et al. (2008) RegulonDB (version 6.0): Gene regulation model of foundation for DNA supercoiling. Proc Natl Acad Sci USA 104:4309–4314. Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters 12. Maurer S, Fritz J, Muskhelishvili G (2009) A systematic in vitro study of nucleoprotein and Textpresso navigation. Nucleic Acids Res 36(Database issue):D120–D124. complexes formed by bacterial nucleoid-associated proteins revealing novel types of 35. Jeong KS, Ahn J, Khodursky AB (2004) Spatial patterns of transcriptional activity in DNA organization. J Mol Biol 387:1261–1276. the chromosome of Escherichia coli. Genome Biol 5:R86. 13. Colland F, Barth M, Hengge-Aronis R, Kolb A (2000) Sigma factor selectivity of 36. Berger M, et al. (2010) Coordination of genomic structure and transcription by the Escherichia coli RNA polymerase: Role for CRP, IHF and lrp transcription factors. EMBO main bacterial nucleoid-associated protein HU. EMBO Rep 11:59–64. J 19:3028–3037. 37. Fuller RS, Kornberg A (1983) Purified dnaA protein in initiation of replication at the 14. Shin M, et al. (2005) DNA looping-mediated repression by histone-like protein H-NS: Escherichia coli chromosomal origin of replication. Proc Natl Acad Sci USA 80: Specific requirement of Esigma70 as a cofactor for looping. Genes Dev 19:2388–2398. 5817–5821. 15. Maurer S, Fritz J, Muskhelishvili G, Travers A (2006) RNA polymerase and an activator 38. Tani TH, Khodursky A, Blumenthal RM, Brown PO, Matthews RG (2002) Adaptation to form discrete subcomplexes in a transcription initiation complex. EMBO J 25: famine: A family of stationary-phase genes revealed by microarray analysis. Proc Natl 3784–3790. – 16. Blot N, Mavathur R, Geertz M, Travers A, Muskhelishvili G (2006) Homeostatic Acad Sci USA 99:13471 13476. regulation of supercoiling sensitivity coordinates transcription of the bacterial 39. Dorman CJ (2004) H-NS: A universal regulator for a dynamic genome. Nat Rev – genome. EMBO Rep 7:710–715. Microbiol 2:391 400. 17. Sonnenschein N, Geertz M, Muskhelishvili G, Hütt MT (2011) Analog regulation of 40. Valens M, Penaud S, Rossignol M, Cornet F, Boccard F (2004) Macrodomain – metabolic demand. BMC Syst Biol 5:40. organization of the Escherichia coli chromosome. EMBO J 23:4330 4341. 18. Ball CA, Osuna R, Ferguson KC, Johnson RC (1992) Dramatic changes in Fis levels upon 41. Marr C, Geertz M, Hütt MT, Muskhelishvili G (2008) Dissecting the logical types of fi nutrient upshift in Escherichia coli. J Bacteriol 174:8043–8056. network control in gene expression pro les. BMC Syst Biol 2:18. 19. Schneider R, Travers A, Kutateladze T, Muskhelishvili G (1999) A DNA architectural 42. Ferrándiz MJ, Martín-Galiano AJ, Schvartzman JB, de la Campa AG (2010) The protein couples cellular physiology and DNA topology in Escherichia coli. Mol genome of Streptococcus pneumoniae is organized in topology-reacting gene Microbiol 34:953–964. clusters. Nucleic Acids Res 38:3570–3581. 20. Keane OM, Dorman CJ (2003) The gyr genes of Salmonella enterica serovar 43. Hardy CD, Cozzarelli NR (2005) A genetic selection for supercoiling mutants of Typhimurium are repressed by the factor for inversion stimulation, Fis. Mol Genet Escherichia coli reveals proteins implicated in chromosome structure. Mol Microbiol Genomics 270:56–65. 57:1636–1652. 21. Weinstein-Fischer D, Altuvia S (2007) Differential regulation of Escherichia coli 44. Montero Llopis P, et al. (2010) Spatial organization of the flow of genetic information topoisomerase I by Fis. Mol Microbiol 63:1131–1144. in bacteria. Nature 466:77–81. 22. Claret L, Rouvière-Yaniv J (1996) Regulation of HU α and HU β by CRP and FIS in 45. Rochman M, Aviv M, Glaser G, Muskhelishvili G (2002) Promoter protection by Escherichia coli. J Mol Biol 263:126–139. a transcription factor acting as a local topological homeostat. EMBO Rep 3:355–360. 23. Falconi M, Brandi A, La Teana A, Gualerzi CO, Pon CL (1996) Antagonistic involvement 46. Condon C, Liveris D, Squires C, Schwartz I, Squires CL (1995) rRNA operon multiplicity of FIS and H-NS proteins in the transcriptional control of hns expression. Mol in Escherichia coli and the physiological implications of rrn inactivation. J Bacteriol Microbiol 19:965–975. 177:4152–4156.

Sobetzko et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | E49 Downloaded by guest on September 27, 2021 47. Cabrera JE, Jin DJ (2003) The distribution of RNA polymerase in Escherichia coli is 62. Levanon SS, San KY, Bennett GN (2005) Effect of oxygen on the Escherichia coli ArcA dynamic and sensitive to environmental cues. Mol Microbiol 50:1493–1505. and FNR regulation systems and metabolic responses. Biotechnol Bioeng 89:556–564. 48. Dong H, Nilsson L, Kurland CG (1996) Co-variation of tRNA abundance and codon 63. Mika F, Hengge R (2005) A two-component phosphotransfer network involving ArcB, usage in Escherichia coli at different growth rates. J Mol Biol 260:649–663. ArcA, and RssB coordinates synthesis and proteolysis of sigmaS (RpoS) in E. coli. Genes 49. Lau IF, et al. (2003) Spatial and temporal organization of replicating Escherichia coli Dev 19:2770–2781. – chromosomes. Mol Microbiol 49:731 743. 64. Zechiedrich EL, Khodursky AB, Cozzarelli NR (1997) Topoisomerase IV, not gyrase, 50. Adachi S, Fukushima T, Hiraga S (2008) Dynamic events of sister chromosomes in the decatenates products of site-specific recombination in Escherichia coli. Genes Dev 11: cell cycle of Escherichia coli. Genes Cells 13:181–197. 2580–2592. 51. Kim J, et al. (2004) Fundamental structural units of the Escherichia coli nucleoid 65. Zechiedrich EL, et al. (2000) Roles of topoisomerases in maintaining steady-state DNA revealed by atomic force microscopy. Nucleic Acids Res 32:1982–1992. – 52. Frenkiel-Krispin D, et al. (2004) Nucleoid restructuring in stationary-state bacteria. supercoiling in Escherichia coli. J Biol Chem 275:8103 8113. Mol Microbiol 51:395–405. 66. Jishage M, Ishihama A (1998) A stationary phase protein in Escherichia coli with 53. Olliver A, Saggioro C, Herrick J, Sclavi B (2010) DnaA-ATP acts as a molecular switch to binding activity to the major sigma subunit of RNA polymerase. Proc Natl Acad Sci control levels of ribonucleotide reductase expression in Escherichia coli. Mol Microbiol USA 95:4953–4958. 76:1555–1571. 67. Robbe-Saule V, et al. (2006) Physiological effects of crl in Salmonella are modulated 54. Travers AA, Kari C, Mace HAF (1981) Transcriptional regulation by bacterial RNA by SigmaS and promoter activity. J Bacteriol 189:2976–2987. polymerase. Genetics as a Tool in Microbiology, SGM Symposia, eds Glover S, 68. Stepanova E, et al. (2007) Analysis of promoter targets for Escherichia coli Hopwood D (Cambridge Univ Press, Cambridge, UK), Vol XXXI, pp 111–130. transcription elongation factor GreA in vivo and in vitro. J Bacteriol 189:8772–8785. 55. Wiggins PA, Cheveralls KC, Martin JS, Lintner R, Kondev J (2010) Strong intranucleoid 69. Susa M, Kubori T, Shimamoto N (2006) A pathway branching in transcription interactions organize the Escherichia coli chromosome into a nucleoid filament. Proc initiation in Escherichia coli. Mol Microbiol 59:1807–1817. Natl Acad Sci USA 107:4991–4995. 70. Potrykus K, et al. (2006) Antagonistic regulation of Escherichia coli ribosomal RNA fl 56. Af erbach H, Schröder O, Wagner R (1998) Effects of the Escherichia coli DNA- rrnB P1 promoter activity by GreA and DksA. J Biol Chem 281:15238–15248. – binding protein H-NS on rRNA synthesis in vivo. Mol Microbiol 28:641 653. 71. Rutherford ST, et al. (2007) Effects of DksA, GreA, and GreB on transcription 57. Spurio R, et al. (1992) Lethal overproduction of the Escherichia coli nucleoid protein initiation: Insights into the mechanisms of factors that bind in the secondary channel H-NS: Ultramicroscopic and molecular autopsy. Mol Gen Genet 231:201–211. of RNA polymerase. J Mol Biol 366:1243–1257. 58. Scolari VF, Bassetti B, Sclavi B, Lagomarsino MC (2011) Gene clusters reflecting 72. Blankschien MD, et al. (2009) TraR, a homolog of a RNAP secondary channel macrodomain structure respond to nucleoid perturbations. Mol Biosyst 7:878–888. interactor, modulates transcription. PLoS Genet 5:e1000345. 59. Dong T, Schellhorn HE (2009) Control of RpoS in global gene expression of Escherichia 73. Paul BJ, et al. (2004) DksA: A critical component of the transcription initiation coli in minimal media. Mol Genet Genomics 281:19–33. 60. Gao F, Zhang CT (2007) DoriC: A database of oriC regions in bacterial genomes. machinery that potentiates the regulation of rRNA promoters by ppGpp and the – Bioinformatics 23:1866–1867. initiating NTP. Cell 118:311 322. 61. Alexeeva S, Hellingwerf KJ, Teixeira de Mattos MJ (2003) Requirement of ArcA for 74. Lemke JJ, et al. (2011) Direct regulation of Escherichia coli ribosomal protein redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic promoters by the transcription factors ppGpp and DksA. Proc Natl Acad Sci USA 108: conditions. J Bacteriol 185:204–209. 5712–5717.

E50 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al. Downloaded by guest on September 27, 2021